Many suffer debilitating nerve injuries each year and the majority will not achieve functional recovery. Nerve regeneration requires the (1) robust and (2) directed extension of regenerating processes through the injury site to quickly reconnect with proper targets. The failure of the local glial cellsperipheral nerve support cells, Schwann cells, to migrate into and re-populate the injury site has been identified to be a limiting step following large-gap peripheral nerve injury19-21. It is well understood that Schwann cells can promote neuronal re-growth22-25 and our lab has quantitatively demonstrated that local Schwann cell orientation can also direct local neurite outgrowth3. We hypothesize that uniaxially oriented Schwann cells would serve to both promote and efficiently direct neurite outgrowth through an injury site. Preliminary work in this proposal utilizes physiologically-compatible, low level electric stimulation to induce aligned groups of Schwann cells ("cords"). Electric stimulation represents one means to control cell alignment, but the mechanism underlying alignment by electric stimulation remains unknown. Preliminary work also demonstrates a 2-fold increase in outgrowth for neurons co-cultured with electrically-stimulated Schwann cells relative to co-culture with unstimulated cells. The basis of this robust neuronal growth, mediated by the electrically-stimulated Schwann cells, also remains unknown. Therefore, the goals of the proposal are 1) to use electrical stimulation to enhance the Schwann cell phenotype and control Schwann cell alignment within a 3D scaffold as well as 2) determine the mechanistic basis by which these biophysical forces impact both Schwann cells and neurons to promote extensive and directed neurite outgrowth via the following aims: Aim 1: Determine the optimal parameters to obtain highly-oriented Schwann cell cords within a 3D composite hydrogel using physiologically-compatible pulsed DC electrical stimulation. Aim 2: Determine if neurite outgrowth is enhanced in response to stimulated and oriented Schwann cell cords developed in Aim 1. Aim 3: Identify the mechanisms underlying the alignment of Schwann cells by electrical stimulation. Aim 4: Identify the mechanisms underlying the increased neurite outgrowth by the aligned Schwann cells. If successful, this proposed research will (1) Identify features of the electrical stimulation responsible for the observed changes in morphology and phenotype to motivate the rational in vivo presentation of this biophysical force. (2) Identify the mechanistic basis for electrically-mediated Schwann cell changes which direct and promote robust neurite outgrowth. Understanding the aforementioned mechanism, would serve to (3) demonstrate the utility of electrical stimulation to synergistically enhance Schwann cells for the rapid and robust axonal guidance relevant to both the fields of Neuroscience and Neural Engineering or (4) provide targets to initiate these changes chemically in the absence of electrical stimulation. PUBLIC HEALTH RELEVANCE: Schwann cell (SC) participation is key participant supporting re-growth following peripheral nerve injury. The goal of this work is to utilize electrical stimulation to control SC alignment and enhance SC phenotype and identify the mechanistic basis for these electrically-induced changes. These aligned SC's serve to 1) promote and 2) direct robust neurite outgrowth. Mechanistic understanding of electrically-mediated changes will aid in the rational presentation of electric stimulation relevant to Neuroscience and Tissue Engineering.